ABTS底物检测漆酶活力条件和算法比较——以长白山两种林分土壤为例

Comparisons of Experiment Conditions and Calculation Methods for Laccase Activity Detection with ABTS-A Case Study with 2 Forest Stands in Changbai Mountain

选取长白山针叶林与阔叶林A层土壤,以0.25~12.5 mmol L^(-1)不同终浓度2,2'-连氮-双(3-乙基苯并噻唑-6-磺酸)(ABTS)为底物,考察漆酶氧化产物在0~32、0~54和0~95 min吸光值线性变化率,并藉此获得3个不同时间段线性拟合漆酶活力值;以一级反应动力学方程拟合最大吸光值增幅及相应平均反应时间,计算吸光值平均变化率,并藉此获得非线性拟合漆酶活力值。当ABTS浓度为7.5 mmol L^(-1)时,反应体系吸光值增幅和酶活最大。阔叶林反应体系吸光值增量显著大于针叶林,前者在0~300 min持续增加,且不同算法间漆酶活力差异不显著;后者仅在0~21 min显著变化,且非线性拟合漆酶活力值显著高于线性拟合。非线性拟合测算的漆酶活力变异系数整体上低于线性拟合。因此,森林土壤漆酶活力测定应重视酶底物浓度、反应时间、算法和林分条件变化带来的显著影响。

Soil samples were separately collected from the coniferous and broad-leaved forest ecosystems in Changbai Mountain. The 2,2＇-azinobis （3-ethyl-benzthiazoline-6-sulfonic acid） （i.e. ABTS） was used as substrate at the final concentrations of 0.25, 0.5, 1.25, 2, 3, 5, 7.5 and 12.5 mmol L-1, respectively. In 3 selected time spans, i.e. 0 - 32, 0 - 54 and 0 - 95 min, we firstly used SLOPE function to calculate the increased absorbance of ABTS oxidation across the above incubation time spans, and then separately assessed the linearly derived laccase activities within 0 - 32 min （i.e. L32）, 0 - 54 min （i.e. L-1） and 0 - 95 min （i.e. L95）. Simultaneously, using first-order kinetic equation, we derived the maximal increased absorbance of ABTS oxidation and mean reaction time, thereafter estimated the mean increased absorbance of ABTS oxidation during 0 - 300 min and the non-linear laccase activities （i.e. LN）. The results showed that the maximal increment of absorbance of ABTS oxidation and laccase activities were observed at the final ABTS concentration of 7.5 mmol L-l. The broad-leaved forest stand presented greater increment of absorbance of ABTS oxidation compared to the coniferous forest stand. Particularly, in the broad-leaved forest stand, the absorbance of ABTS oxidation continuously increased during 0 - 300 min and the laccase activity values had no significant difference between L32, Ls4, L95 and LN. However, as for the coniferous stand, the significant increase of absorbance was only observed within 0 - 21 min but not later on, and the laccase activity values were significantly greater with LN than those of L32, L-1 and L95. The coefficient of variation than the linear ones. We conclude that the forest （CV） between different substrates was on average lower with LN soil laccase detection is substantially determined by ABTS concentration, reaction time, calculating method and forest stand.